A Pseudomonas aeruginosa mutant derived by random mutagenesis with N-methyl-N¢-nitro-N-nitrosoguanidine, producing high levels of the rhamnolipid biosurfactants was selected on Siegmund-Wagner (SW) plates. The mutant designated P. aeruginosa PTCC1637 produces rhamnolipids at concentrations 10 time more than parent strain. NMR analysis and surface tension measurement showed that the biosurfactants produced by the mutant were identical to those produced by the wildtype strain. The biosurfactants exhibited a low surface tension of 28.0 mN m-1 and a low critical micelle concentration of 9 mg l-1. Similar to the wildtype strain, the mutant produced biosurfactants at the stationary phase